Vitamin A distribution and content in tissues of the lamprey Lampetra japonica.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 276A:134 –142 (2004) Vitamin A Distribution and Content in Tissues of the Lamprey, Lampetra japonica HEIDI L. WOLD,1 KENJIRO WAKE,2 NOBUYO HIGASHI,3 DAREN WANG,3 NAOSUKE KOJIMA,3 KATSUYUKI IMAI,3 RUNE BLOMHOFF,1 AND HARUKI SENOO3* 1 Institute for Nutrition Research, Faculty of Medicine, University of Oslo, Oslo, Norway 2 Department of Anatomy, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo, Japan 3 Department of Anatomy, Akita University School of Medicine, Akita, Japan ABSTRACT Vitamin A (retinol and retinyl ester) distribution and content in tissues of a lamprey (Lampetra japonica) were analyzed by morphological methods, namely, gold chloride staining, ﬂuorescence microscopy to detect speciﬁc vitamin A autoﬂuorescence, and electron microscopy, as well as high-performance liquid chromatography (HPLC). Hepatic stellate cells showed an abundance of vitamin A stored in lipid droplets in their cytoplasm. Similar cells storing vitamin A were present in the intestine, kidney, gill, and heart in both female and male lampreys. Morphological data obtained by gold chloride staining method, ﬂuorescence microscopy, transmission electron microscopy, and HPLC quantiﬁcation of retinol were consistent. The highest level of total retinol measured by HPLC was found in the intestine. The second and third highest concentrations of vitamin A were found in the liver and the kidney, respectively. These vitamin A-storing cells were not epithelial cells, but mesoderm-derived cells. We propose as a hypothesis that these cells belong to the stellate cell system (family) that stores vitamin A and regulates homeostasis of the vitamin in the whole body in the lamprey. Fibroblastic cells in the skin and somatic muscle stored little vitamin A. These results indicate that there is difference in the vitamin A-storing capacity between the splanchnic and intermediate mesoderm-derived cells (stellate cells) and somatic and dorsal mesoderm-derived cells (ﬁbroblasts) in the lamprey. Stellate cells derived from the splanchnic and intermediate mesoderm have high capacity and ﬁbroblasts derived from the somatic and dorsal mesoderm have low capacity for the storage of vitamin A in the lamprey. Anat Rec Part A 276A:134 –142, 2004. © 2004 Wiley-Liss, Inc. Key words: vitamin A; lamprey; stellate cell; gold chloride; ﬁbroblast; mesoderm Vitamin A is involved in many biological processes such as cell growth, differentiation, morphogenesis, and apoptosis (Blomhoff, 1994). Homeostasis of vitamin A is rigidly controlled in the body (Blomhoff et al., 1990) with hepatic stellate cells (vitamin A-storing cells, fat-storing cells, interstitial cells, Ito cells) playing pivotal roles in its regulation (Wake, 1980; Senoo et al., 1984, 1997, 1998; Wake and Senoo, 1986; Blomhoff and Wake, 1991; Senoo and Hata, 1994; Senoo, 2004). In the liver of vertebrates, vitamin A (retinol and retinyl esters) is stored in hepatic stellate cells that were ﬁrst described by von Kupffer (1876) and Rothe (1882), who used the classical gold chloride method (Wake, 1964, 1971, 1974, 1980, 1982; Wake et al., 1986). The stellate cells are distributed in the space between the sinusoidal endothelial cells and the hepatic parenchymal cells and contain characteristically a number of lipid droplets that store retinol in the form of retinyl esters (Higashi and Senoo, 2003; Sato et al., 2003). These lipid droplets emit retinol autoﬂuorescence under a ﬂuorescence microscope and are © 2004 WILEY-LISS, INC. stained black by the gold chloride method. Storage of retinol in these cells has been also conﬁrmed by means of Grant sponsor: Ministry of Education, Culture, Sports, Science, and Technology of Japan; Grant numbers: Grant-in-Aid for Scientiﬁc Research (B) (2) (13470001), Grant-in-Aid for Scientiﬁc Research (C) (11670001), Grant-in-Aid for Exploratory Research (15659418). Heidi L. Wold and Kenjiro Wake equally contributed to this study. The present address of Kenjiro Wake: Liver Research Unit, Minophagen Pharmaceutical Co. Ltd., No. 3 Tomizawa Bldg. 4F, 3-2-7 Yotsuya, Shinjuku-ku, Tokyo 160-0004, Japan. *Correspondence to: Haruki Senoo, Department of Anatomy, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan. Fax: ⫹81-18-834-7808. E-mail: email@example.com Received 18 September 2002; Accepted 2 September 2003 DOI 10.1002/ar.a.10345 VITAMIN A DISTRIBUTION AND CONTENT IN LAMPREY 3 radioautography after administration of H-retinol (Hirosawa and Yamada, 1973). Stellate cells are distributed not only in the liver but also in other organs (Kusumoto and Fujita, 1977; Wake, 1980; Nagy et al., 1997; Matano et al., 1999); however, the exact distribution and the content of vitamin A in each organ in the whole human body are still unknown. It is of interest to know how organisms have developed mechanisms to utilize, regulate, and store retinol during vertebrate evolution. The lamprey is an intriguing model for analyzing the evolution of retinol in the whole body of a vertebrate, because due to its lowly position on the taxonomic scale of vertebrates, and the fact that the lamprey can claim as its ancestors the primitive ostracoderms, the lamprey has been of interest to investigators studying both ontogenetic and phylogenetic development (Youson, 1985). By the Carr-Price method a large amount of retinol was demonstrated in the body of the adult lamprey (Lampetra japonica) during its spawning migration (Higashi et al., 1958). In addition, the appearance of retinol in lamprey tissues was shown by Higashi and Yamada (1962) by use of ﬂuorescence microscopy. The distribution, localization, and ﬁne structure of the stellate cells in the liver of adult lamprey were studied by Youson et al. (1985, 1987), Peek et al. (1979), and Wake et al. (1987). The stellate cells in the lamprey liver store vitamin A as lipid droplets in their cytoplasm, but differ in some of their properties from their counterpart in the mammalian liver. These cells are responsible for periductal ﬁbrosis during biliary atresia in the lamprey (Yamamoto et al., 1986; Youson et al., 1987). Pillar cells in gill ﬁlaments (Wake et al., 1989) and mesangial cells in the renal corpuscle (Bauer and Wake, 1996) in the lamprey also store vitamin A. However, the distribution and exact quantiﬁcation and qualiﬁcation of vitamin A (retinol, retinyl esters with fatty acids) in other tissues and organs in the whole body of the lamprey have not yet been thoroughly investigated. Therefore, to extend previous reports and analyze further the distribution of stellate cells and the vitamin A content in organs in the whole body and in serum of the lamprey, we performed the present study by using high-performance liquid chromatography (HPLC). MATERIALS AND METHODS Adult lampreys (Lampetra japonica), classiﬁed as stage VII in the life cycle of the anadromous parasitic lamprey according to the criteria deﬁned by Hardisty and Potter (1971), were caught in rivers in Akita Prefecture, Japan, in January and February 2000 –2002. A total of 11 animals (5 females and 6 males) were investigated in the present study. The protocol for animal experimentation described in this paper was previously approved by the Animal Research Committee of Akita University School of Medicine. All subsequent animal experiments adhered to the Guidelines for Animal Experimentation of the university. MORPHOLOGICAL METHODS In total, seven lampreys (three females and four males) were prepared for the morphological analysis. Four animals (two females and two males) were anesthetized in a 0.01% freshwater solution of meta-aminobezoic acid eth- 135 ylester methanesulfonate (MS222, Sankyo, Tokyo, Japan). After deep anesthetization, each organ (liver, kidney, heart, intestine, gill, somatic muscle, and skin) was taken from the animals. The rest of the body was then immediately frozen at – 80°C. Gold Chloride Staining Blocks of fresh organs from four lampreys (two females and two males) were subjected to the gold chloride staining method modiﬁed by Wake (Wake, 1971; Wake et al., 1986, 1987). Brieﬂy, the tissue blocks were immersed in 0.05% chromic acid for about 2 hr at room temperature. The blocks were then frozen and cut into 50-m-thick sections and placed in 0.05% chromic acid for 10 min. Then they were incubated in the gold chloride solution (1 ml of 1% gold chloride, 1 ml of 1% HCl, 98 ml of distilled water) at 18 –20°C in total darkness for 17–20 hr. When the sections had turned red-purple, they were dehydrated with ethanol and mounted in balsam. The sections were examined under a light microscope, and color photomicrographs were taken with Ektachrome ASA 100 ﬁlm (Eastman Kodak). Also, monochromic photomicrographs were taken by using Panatomic-X ASA 32 ﬁlm (Eastman Kodak). Fluorescence Microscopy Other tissue blocks were immersed in 3.7% formaldehyde for 24 hr at 4°C in total darkness, and 10-m-thick sections were prepared with a freezing microtome as described previously (Wake, 1971; Senoo and Wake, 1985; Wake et al., 1986; Nagy et al., 1997). The sections were examined under a Zeiss Axioskop (excitation ﬁlter BP365/ 12, barrier ﬁlter BP495/40) for the detection of the rapidly fading green autoﬂuorescence characteristic of vitamin A. Color photomicrographs were taken by using Ektachrome ASA 400 ﬁlm (Eastman Kodak), and monochromic photomicrographs by using Tri-X Pan ASA 400 ﬁlm (Eastman Kodak). Other tissue blocks ﬁxed in 3.7% formaldehyde were dehydrated in a graded ethanol series, embedded in parafﬁn, and sectioned. Sections were stained with hematoxylin and eosin, examined under a light microscope, and photographed by using monochromatic Panatomic-X ASA 32 ﬁlm (Eastman Kodak). Transmission Electron Microscopy The organs except the liver from three animals (one female and two males) were perfused in situ for 1 or 2 min via the heart with 1.5% glutaraldehyde and 0.062 M cacodylate buffer, pH 7.4, containing 1% sucrose. Part of the excised liver was divided into blocks and perfused for 1 or 2 min by injection of the ﬁxatives through branches of blood vessels whose lumens appeared on the cut surface of the blocks as described previously (Wake et al., 1987). Under a dissection microscope the perfusion was performed with a syringe ﬁtted with a needle (26 G ⫻ 1/2⬙). The ﬁxative consisted of 1.5% glutaraldehyde and 0.062 M cacodylate buffer, pH 7.4, containing 1% sucrose. After the perfusion ﬁxation the organs were cut into small blocks, and then the small tissue blocks were postﬁxed in 2% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4, for 2 hr at 4°C, dehydrated in a graded ethanol series, and embedded in Poly/Bed 812. Ultrathin sections were cut on an ultramicrotome and stained with 7% uranyl acetate 136 WOLD ET AL. and 0.4% lead citrate. Thin sections were examined with a JEOL-100 CX electron microscope (JEOL, Tokyo, Japan) at an acceleration voltage of 100 kV. Thick sections were examined under a light microscope after staining with 1% toluidine blue containing 1% borax. ANALYSIS OF RETINOL AND RETINYL ESTERS BY HPLC The content of vitamin A in serum (collected from the incised gill), heart, liver, intestine, gonads, kidney, gill, eye, brain, and somatic muscle obtained from the remaining four lampreys (two females and two males) was analyzed by HPLC. Both retinyl esters (palmitate, stearate, oleate, and linoleate) and retinol were quantiﬁed. Retinyl esters were extracted according to Barua et al. (1993) with some minor modiﬁcations. Tissues (5–10 mg of each) were homogenized in 90 l of ice-cold phosphatebuffered saline (PBS) before the addition of 400 l of ice-cold 2-propanol/acetone (50:50 v/v). This mixture was then vigorously shaken for 5 min and centrifuged for 15 min at 3,500 g at 10°C. An aliquot of 80 l was injected into an HPLC system equipped with a 250 ⫻ 4.6 mm C30 column from YMC (Midford, MA) and using an acetonitrile/dichloromethane (70:30 v/v) mobile phase delivered at 2 ml/min (Furr et al., 1986). For serum analysis, 900 l of 2-propanol was added to 300 l of serum. After shaking and centrifugation as described above, an aliquot of 100 l was injected into an HPLC system equipped with a 250 ⫻ 2.0 mm C18 column from Merck and the same mobile phase as used for tissues delivered at 0.25 ml/min. For retinol analyses, 50 mg of tissue was homogenized in 450 l of ice-cold PBS before the addition of 2,000 l of ice-cold 2-propanol containing all-trans-9-(4-methoxy2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nonatetraen1-ol (TMMP-retinol) as an internal standard and butylated hydroxytoluene (BHT) (10 mg/l) as an antioxidant (for serum, 400 l ⫹ 1,200 l 2-propanol containing TMMP-retinol and BHT). After shaking and centrifugation as described above, an aliquot of 1,000 l was injected into the HPLC system combining on-line solid-phase extraction and column switching (Gundersen and Blomhoff, 1998). Mobile phases M1 (2.2 ml/min), M2 (1 ml/min), and M3 (0.5 ml/min) from pumps 1, 2, and 3 were 100% water, 100% methanol, and acetonitrile/water (85:15 v/v), respectively. Both retinyl esters and retinol were detected at 325 nm. All analyses were performed in duplicate from each sample. RESULTS External Morphology The head and associated portion of the adult lamprey (Lampetra japonica) in lateral view showed the morphology typical of this species (Fig. 1). Light and Electron Microscopy Liver. In contrast to the larval lamprey (ammocoete) and all other vertebrates, the adult lamprey does not have a bile duct and the associated storage chamber, the gall bladder (Sterling et al., 1967; Youson and Sidon, 1978; Youson, 1981c; Sidon and Youson, 1983). The adult lamprey liver contains both parenchymal cells and nonparenchymal cells, namely, sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells (vitamin A-storing cells) (Youson et al., 1985). The stellate cells are located not only perisinusoidally, but also perivascularly, and are also found in the subcapsular dense connective tissue. Under the ﬂuorescence microscope, rapid-fading green autoﬂuorescence of vitamin A emanated from cells among the liver parenchyma and within extensive areas of the interstitial connective tissue (Fig. 2). The ﬂuorescing cells in the parenchyma were not parenchymal cells, but hepatic stellate cells. In the interstitial connective tissue the ﬂuorescing cells were ﬁbloblast-like cells. The cell density of ﬂuorescing cells was higher in the connective tissue than among the parenchymal cells. These ﬂuorescing cells were speciﬁcally stained black by the gold chloride method (Fig. 3). Collagen ﬁbers were stained red by this method. Thus, the gold chloride method speciﬁcally stained the vitamin A-storing cells black. Gill. The lamprey gill is a respiratory organ. Blood pumped by the heart ﬂows through the ventral aorta into afferent brachial arteries supplying capillaries in the gill lamellae and then into the efferent brachial arteries, which join to form the dorsal aorta (Randall, 1972). Only the pillar cells in the gill emitted the autoﬂuorescence of vitamin A detected under the ﬂuorescence microscope (Fig. 4). These cells were regularly distributed in the gill ﬁlaments. None of the epithelial cells lining the external surface of the gill showed the vitamin A autoﬂuorescence. Kidney. The elements of the adult opisthonephroi are conﬁned within the wedge-shaped posterior portion of the paired nephric folds, which extend into the coelomic cavity from a dorsal mass of adipose tissue and pigment cells (Youson, 1981b). The three major components of the opisthonephroi are the tubules, the renal corpuscle, and the archinephric duct. The most conspicuous feature of the adult opisthonephros, and a feature that distinguishes it from the adult kidneys of other vertebrates, is the presence of a large, usually single, compound renal corpuscle in each kidney. Each kidney is composed of a single glomus or network of capillaries that extends almost the entire length of the kidney (Youson, 1981b). Throughout the length of the renal corpuscle, tubules radiate out in a semicircle from their origin at the nephric capsules. Each tubule can be divided into several speciﬁc regions or segments that have distinct morphological features (Youson, 1981b). Mesangial cells within the renal corpuscle and cells in the interstitium around the renal tubules emitted vitamin A autoﬂuorescence (Fig. 5); however, no epithelial cells of the renal corpuscle or tubules showed the autoﬂuorescence. The gold chloride method also demonstrated a speciﬁc reaction of vitamin A in the connective tissue around the renal tubules (Fig. 6), but not in the renal epithelium. The ﬂuorescing cells around the renal tubules were identical with the cells stained black by the gold chloride method. An electron micrograph of a part of the renal corpuscle showed the presence of vitamin A-lipid droplets in the cytoplasm of mesangial cells (Fig. 7). Nuclei of the mesangial cells were deeply indented by the lipid droplets. Neither podocytes nor endothelial cells in the renal corpuscle contained vitamin A-lipid droplets in their cytoplasm. Cells in the interstitial tissue around the renal tubules of the kidney contained vitamin A-lipid droplets in their cytoplasm (Fig. 8). However, no epithelial cells of the renal VITAMIN A DISTRIBUTION AND CONTENT IN LAMPREY Fig. 1. Head and associated portion of a lamprey, Lampetra japonica, in lateral aspect. The lamprey was anesthetized as described in Materials and Methods. Fig. 2. Vitamin A autoﬂuorescence released from stellate cells in the liver parenchyma (arrow) and the interstitial connective tissue (CT). The liver was prepared for ﬂuorescence microscopy as described in Materials and Methods. Bar ⫽ 100 m. Fig. 3. Gold chloride staining of a lamprey liver that reduced gold precipitates speciﬁcally in the stellate cells (arrows). Collagen ﬁbers (CF) are stained red. The liver was prepared for the gold chloride staining method as described in Materials and Methods. Bar ⫽ 10 m. 137 Fig. 4. Vitamin A autoﬂuorescence emitted from clusters of lipid droplets in pillar cells of a gill ﬁlament, as detected by ﬂuorescence microscopy. C, capillary; E, epithelium of a gill ﬁlament. Bar ⫽ 5 m. Fig. 5. Vitamin A autoﬂuorescence is observed in the renal corpuscle (G) and peritubular connective tissue in the kidney. RT, renal tubules. Bar ⫽ 10 m. Fig. 6. Selectively black-stained cells are detected in the peritubular connective tissue by the gold chloride staining method. RT, renal tubules. Bar ⫽ 10 m. 138 WOLD ET AL. Fig. 7. An electron micrograph of a part of the renal corpuscle of a lamprey showing the presence of vitamin A-lipid droplets in the cytoplasm of mesangial cells (M). Nuclei of mesangial cells are deeply indented by the lipid droplets. The kidney was prepared for transmission electron microscopy as described in Materials and Methods. E, endothelial cell; P, podocyte. Bar ⫽ 5 m. Fig. 8. An electron micrograph showing cells storing lipid droplets (arrows) in the peritubular connective tissue of the kidney. EP, epithelial cells of the renal tubule. Bar ⫽ 5 m. Fig. 9. Vitamin A autoﬂuorescence (arrows) is detected in the lamprey heart by ﬂuorescence microscopy. M, heart muscular ﬁber. Bar ⫽ 5 m. Fig. 10. A cell (SC) is observed by transmission electron microscopy to contain a large vitamin A-lipid droplet (A) associated with collagen ﬁbers (CF) in the subendocardial connective tissue of the lamprey heart. E, endothelium; M, heart muscle cell. Bar ⫽ 1 m. tubules stored vitamin A in their cytoplasmic lipid droplets. cence of vitamin A (Fig. 9). These cells were ﬁbroblast-like cells with a morphology different from that of cardiac muscle cells forming muscle ﬁbers. These cells contained one or two large vitamin A-lipid droplets (Fig. 10), whereas the muscle cells contained myoﬁbrils. No endothelial cells in the cardiac muscle contained vitamin Alipid droplets. Heart. The heart of the lamprey consists of a sinus venosus, atrium (auricle), ventricle, and bulbus arteriosus (Fänge, 1972). Histological and electron microscopical studies have shown striated myocardial ﬁbers similar to those of other vertebrates (Fänge, 1972; Hardisty and Rovainen, 1982). Under the ﬂuorescence microscope, cells in the heart emitted the rapid-fading green autoﬂuores- Intestine. The intestine of all species of lampreys begins at its junction with the esophagus near the anterior VITAMIN A DISTRIBUTION AND CONTENT IN LAMPREY 139 margin of the liver and is composed of anterior and posterior parts (Youson, 1981a). In the wall of the intestine, a thin submucosa is sandwiched between the muscle and a lamina propria mucosae (Youson, 1981a). Intense vitamin A autoﬂuorescence was detected in cells of the lamina propria mucosae in the intestine (Fig. 11). These cells were ﬁbroblastic cells, and no autoﬂuorescence was demonstrated in the epithelial cells covering the lamina propria mucosae. The ﬂuorescing cells reacted with gold chloride and were stained black (data not shown). Gold chloride-positive cells were demonstrated also in the transverse section of the intestine showing a part of the typhlosole (data not shown). Under the electron microscope these cells in the lamina propria mucosae that emitted autoﬂuorescence of vitamin A and reacted with gold chloride contained membrane-bound and non-membranebound lipid droplets (Wake, 1974) in their cytoplasm (Fig. 12). Vitamin A autoﬂuorescence was released also from the layers of smooth muscle in the intestine (Fig. 13). These cells reacted with gold chloride and were stained black (data not shown), but they were different from the smooth muscle cells. Skin. The skin of vertebrates is composed of two primary layers, the epidermis and the dermis, which unite to form the peripheral boundary separating the internal milieu of the individual from its external environment. While the epidermis develops from ectodermal tissue and is organized as a stratiﬁed or pseudostratiﬁed epithelium, the dermis is mesodermal in origin and is usually composed of collagen ﬁbers, pigment cells, and elements of the vascular and nervous systems. These dermal components are often separated from the body wall musculature by several rows of fat cells, which constitute the subcutaneous adipose layer (Lethbridge and Potter, 1981). The skin of lampreys consists of epidermis, dermis, and subcutaneous tissue (data not shown). The dermis contains layers of ﬂattened ﬁbroblasts sandwiched between lamellae of collagen bundles. These ﬁbroblasts as well as subcutaneous ﬁbroblasts contained no lipid droplets; no vitamin A was demonstrated in these ﬁbroblasts by either the gold chloride method or ﬂuorescence microscopy (data not shown). Somatic muscle. Striated muscles of the lamprey are classiﬁed as somatic or visceral according to their embryological origins. The somatic muscles, including the extraocular, myotomal, and ﬁn muscles, are derived from the myomeres, whereas the visceral muscles, which are used for feeding and ventilation, arise in the lateral plates of the visceral arches in the head and gill regions (Hardisty and Rovainen, 1982). The myotomes constitute the predominant muscle mass of the lamprey. They retain a clear segmental organization and consist of short, longitudinally orientated striated muscle ﬁbers, both in the body and in the myotomal sheets that cover the visceral muscles in the head and gill regions. The myotomes originate from the myomeres of the somites during embryological development and are thus termed somatic (Hardisty and Rovainen, 1982). Adipose cells loaded with fat droplets in their cytoplasm were intercalated between muscle ﬁbers in the body muscle of the lamprey (data not shown). In gold chloride-stained sections, a small number of tiny gold chloride-reactive cells were interspersed between the muscle ﬁbers (data not shown), but these cells Fig. 11. Vitamin A autoﬂuorescence is detected in cells of the lamina propria (LP). E, epithelium. Bar ⫽ 5 m. Fig. 12. An electron micrograph showing membrane-bound (type I, A1) and non-membrane-bound (type II, A2) lipid droplets (Wake, 1974) in a cell (SC) in the lamina propria of the intestine. NF, unmyelinated nerve ﬁber. Bar ⫽ 1 m. Fig. 13. Vitamin A autoﬂuorescence released from cells in the muscle layers of the intestine. Bar ⫽ 5 m. were different from the adipose cells. By elecron microscopy, small cells that appeared to be identical with the gold chloride-reactive cells present between muscle ﬁbers contained lipid droplets in their cytoplasm (data not 140 WOLD ET AL. TABLE 1. Vitamin A content in tissues of Lampetra japonica analyzed by high-performance liquid chromatography concentration (nmol/g wet tissue)* Female (n ⫽ 2, mean values) Tissue Serum Heart Liver Intestine Gonad Kidney Gill Eye Brain Muscle ROH⫹RE ROH 2 242 558 1687 52 441 278 92a 3a 41 RE 2 0 17 225 88 470 199 1488 10 42 10 431 98 181 28a 54 3a 1 4 7 Male (n ⫽ 2, mean values) %16:0 %18:0 %18:1 %18:2 ROH⫹RE ROH 94 76 81 67 80 67 99 93 100 87 6 1 3 5 0 3 0 0 0 0 0 21 14 25 19 28 1 7 0 13 0 2 2 3 1 2 0 0 0 0 1 127 439 3006 66 231 84 39 6 35 1 7 28 301 31 7 29 10 4 3 RE 0 120 411 2705 35 224 56 29 2 32 %16:0 %18:0 %18:1 %18:2 98 81 85 65 74 64 62 93 100 84 2 0 2 6 0 2 0 0 0 0 0 18 11 26 26 33 37 7 0 16 0 1 2 2 0 1 1 0 0 0 *Retinol (ROH) and retinyl esters (RE) were measured as described in Materials and Methods. The results are presented as mean values. n ⫽ 2 in each group. a n ⫽ 1. 16:0 ⫽ palmitate, 18:0 ⫽ stearate, 18:1 ⫽ oleate, 18:2 ⫽ linoleate. shown), but the size and number of the lipid droplets were small. It was hard to detect autoﬂuorescence of vitamin A in these cells under the ﬂuorescence microscope. Distribution of Vitamin A (Table 1) The content of vitamin A (total retinol), retinol, retinyl ester, and fatty acids (palmitate, stearate, oleate, and linoleate) in various lamprey tissues is presented in Table 1. The highest level of total retinol was found in the intestine. Of the total stored retinol, 88 –90% of it was retinyl esters, mainly retinyl palmitate. The second highest concentration of vitamin A was found in the liver, and 84 – 94% of the total retinol was also in the form of retinyl esters, mainly retinyl palmitate. The third highest level of total retinol was found in the kidney, where 97–98% of it was also retinyl esters, again mainly retinyl palmitate. The levels of total retinol in the heart and gill were also high, but the levels in the somatic muscle and gonads were low, and the levels in the serum and brain were very low. The percentage of retinyl esters to total retinol and the compositions of the four fatty acids, namely, palmitate, stearate, oleate, and linoleate, were essentially the same in both female and male organs examined. Essentially no difference existed in total retinol concentration in each corresponding organ (female liver and male liver, for instance) between female and male lampreys. DISCUSSION The Stellate Cell System Storing Retinol In mammals, hepatic stellate cells store 80% of the total retinol in the whole body as retinyl esters in the lipid droplets in their cytoplasm (Wake, 1971, 1980; Blomhoff et al., 1990; Imai and Senoo, 1998; Imai et al., 2000). Several papers reported that cells in other organs such as kidney, intestine, and pancreas of rats, mice, and humans can store retinol and contribute to the regulation of vitamin A homeostasis (Hirosawa and Yamada, 1973; Wake et al., 1987; Nagy et al., 1997; Matano et al., 1999; Apte et al., 1998; Bachem et al., 1998). To investigate systematically the distribution of cells storing vitamin A in adult lamprey body and to compare this distribution with that of the hepatic stellate cells, we performed this study. Cells responsible for storage of vitamin A were distributed in all the visceral organs examined, namely, intestine, liver, kidney, gill, and heart. The storage cells were not epithelial cells, but mesenchymal cells, and they resembled hepatic stellate cells from the viewpoint of morphology; these cells emitted the autoﬂuorescence of vitamin A, reacted with gold chloride, and ﬁne structure analysis revealed lipid droplets in their cytoplasm. The pillar cells, namely, the lining cells of the blood lacunae in the secondary lamellae of the gill ﬁlaments, have been already described as vitamin A-storing cells of gill ﬁlaments (Wake et al., 1987, 1989). We found lipid droplets containing vitamin A in these cells, which ﬁndings are consistent with these earlier data. Morphological analysis of the kidney indicated that mesangial cells in the renal corpuscle and mesenchymal cells in the interstitium had characteristics of the stellate cell (vitamin A-storing cell). From these data we propose as a hypothesis that the stellate cell system or family exists in splanchnic and intermediate mesoderm-derived tissues in the whole body of the lamprey (Fig. 14). Centralization of the Storage Site for Vitamin A during Evolution Lampreys and hagﬁsh are the only extant jawless ﬁsh or agnathans. They may have shared a common ancestor in the early Cambrian period, but they diverged very early in their evolutionary histories. Lampreys have had a rather conserved evolution since they ﬁrst appeared 350 million years ago (Youson and Al-Mahrouki, 1999). In mammals, vitamin A is mainly stored in the liver; especially hepatic stellate cells store 80% of the vitamin of the whole body. However, vitamin A in the lamprey was found to be localized not only in the hepatic stellate cells, but also in other mesenchymal cells derived from the mesoderm. As mentioned above, we propose a hypothesis that the stellate cell system exists in lampreys. Our present data suggest that the storage site of vitamin A changed from the wide distribution in the splanchnic and intermediate mesoderm-derived tissues in lampreys to the liver in mammals during evolution. This phenomenon VITAMIN A DISTRIBUTION AND CONTENT IN LAMPREY 141 Fig. 14. Distribution of vitamin A in the stellate cell system in the lamprey, Lampetra japonica. Stellate cells (vitamin A-storing cells), derived from the splanchnic and intermediate mesoderm (intestine, liver, kidney, heart, gill), have high capacity; and ﬁbroblasts, derived from the somatic and dorsal mesoderm (skin, somatic muscle), have low capacity for the storage of vitamin A. might be called centralization of the storage site of vitamin A during evolution. Agreement between Morphological and HPLC Analytical Data Morphological data obtained by the gold chloride staining method, ﬂuorescence microscopy, and transmission electron microscopy, and data on retinol quantiﬁed by HPLC were consistent. The high concentration of vitamin A in the intestine detected by HPLC was consistent with the presence of stellate cells that contained large vitamin A-lipid droplets in the intestine. Also, the high concentration of total retinol in the liver found by HPLC was consistent with stellate cells that contained large vitamin A-lipid droplets in hepatic parenchyma and interstitial connective tissue. Thus, the HPLC analysis supported the morphological ﬁndings. Differentiation of Mesodermal Cells We deﬁned the mesenchymal cells containing multiple vitamin A-lipid droplets or a single large vitamin A-lipid droplet as stellate cells, and the mesenchymal cells having no or a single small vitamin A-lipid droplet in their cytoplasm as ﬁbroblasts. Cells belonging to the stellate cell system in the intestine, liver, kidney, heart, and gill in the lamprey stored a large amount of vitamin A. On the other hand, ﬁbroblasts in the skin and somatic muscle stored little vitamin A. These results indicate that the stellate cells, derived from the splanchnic and intermediate mesoderm, have high capacity, and that the ﬁbroblasts, derived from the somatic and dorsal mesoderm, have no or low capacity for the storage of vitamin A. Thus, from the viewpoint of vitamin A storage, there is differentiation of function in mesoderm-derived cells according to their localization in the body (Fig. 14). In conclusion, this is the ﬁrst report of systematic analysis of the stellate cell system in the lamprey, which has shown the difference in vitamin A storage between the stellate cells derived from the splanchnic and intermediate mesoderm and ﬁbroblasts derived from the somatic and dorsal mesoderm. ACKNOWLEDGMENTS The authors thank Dr. Mitsuru Sato (Department of Anatomy, Akita University School of Medicine) for his valuable discussions. Expert technical support by Mitsutaka Miura (Department of Anatomy, Akita University School of Medicine) is also highly appreciated. 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